Multi-layered network structure of amino acid (AA) metabolism characterized by each essential AA-deficient condition

Summary. The concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. In this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid. In the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: 1) Lysine is located at the top control level and has effects on almost all of the other plasma amino acids. 2) Threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. 3) Threonine and methionine are interrelated to each other and form a loop structure.     

低温驯化后水椰八角铁甲生理活性物质含量的变化

【目的】分析低温驯化后水椰八角铁甲Octodonta nipae体内各种生理活性物质含量的变化,为揭示水椰八角铁甲耐寒机制提供理论基础。【方法】以25℃处理为对照,将水椰八角铁甲各虫态于12.5, 15, 17.5和20℃驯化处理10 d后,比较其体内的游离水、蛋白质、氨基酸、粗脂肪、甘油和可溶性糖原等生理活性物质含量的变化。【结果】低温驯化显著影响铁甲体内上述物质的含量水平。低温驯化能提高该铁甲体内甘油和游离氨基酸的含量,降低游离水、蛋白质、粗脂肪、可溶性糖原的含量。与对照相比,12.5℃驯化对该铁甲各虫态体内游离水含量存在显著影响(P<0.01);经12.5, 15, 17.5和20.0℃驯化后,该铁甲各虫态体内蛋白质、粗脂肪含量与对照相比均存在显著差异(P<0.01);经125,150和175℃驯化后,该铁甲各虫态体内游离氨基酸含量与对照存在显著差异(P<0.01);经12.5,15.0,17.5和20℃驯化后,该铁甲各虫态（2龄幼虫除外）体内甘油含量与对照相比存在显著差异(P<0.01);12.5, 15.0, 17.5和20℃驯化对除2龄幼虫、蛹和成虫外的铁甲其他各虫态体内可溶性糖原含量存在显著影响。经15.0℃驯化处理后铁甲各虫态体内平均甘油含量最高,比对照25℃的平均甘油含量高出约9.4倍,而经12.5℃驯化处理的虫体内的平均甘油含量仅增加3.5倍。【结论】低温驯化对水椰八角铁甲体内相关生理活性物质含量的影响效能是有限的;水椰八角铁甲可以根据不良的环境条件调节最佳的生理状况,以适应未来的环境和达到最强的适应性。     

Plasma essential amino acids changes in sea-bass (Dicentrarchus labrax) after feeding diets deficient and supplemented in L-methionine

A fishmeal-based reference diet, a soybean-yeast-based methionine (MET)-deficient diet and two MET-supplemented diets were fed to groups of yearling sea-bass, reared in sea-water at 21 +/- 1 degrees C. Levels of most free essential amino acids (EAA) reached a maximum concentration in plasma between 4 and 8 hr after a single meal. Plasma MET level remained very low with the deficient diet, exhibiting by 4 hr a 10-fold reduction when compared to the reference diet, although MET content was divided only by two in the former. Daily MET intake was then maintained under minimum requirement in fish fed the deficient diet. Supplemented MET reached peak level in plasma substantially sooner than the rest of the EAA, indicating faster absorption and degradation when compared to MET incorporated in feed proteins. Evolution of methionine sulfoxide (MSO) levels in plasma after feeding MET-supplemented diets gives evidences that a degradation pathway of MET into MSO exists in sea-bass.     

The aspartic acid metabolic pathway, an exciting and essential pathway in plants

Summary. Aspartate is the common precursor of the essential amino acids lysine, threonine, methionine and isoleucine in higher plants. In addition, aspartate may also be converted to asparagine, in a potentially competing reaction. The latest information on the properties of the enzymes involved in the pathways and the genes that encode them is described. An understanding of the overall regulatory control of the flux through the pathways is undisputedly of great interest, since the nutritive value of all cereal and legume crops is reduced due to low concentrations of at least one of the aspartate-derived amino acids. We have reviewed the recent literature and discussed in this paper possible methods by which the concentrations of the limiting amino acids may be increased in the seeds.     

Different environmental temperatures affect amino acid metabolism in the eurytherm teleost Senegalese sole (Solea senegalensis Kaup, 1858) as indicated by changes in plasma metabolites

Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26°C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18°C were acclimated to either cold (12°C) or warm (26°C) environmental temperatures for 21?days. Fish maintained at 18°C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26°C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12°C. When sole is acclimated to 26°C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation.     

Indispensable amino acid concentrations decrease in tissues of stomachless fish,common carp in response to free amino acid- or peptide-based diets

Summary. The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.     

Cysteine dioxygenase and γ-glutamylcysteine synthetase activities in primary cultured hepatocytes respond to sulfur amino acid supplementation in a reciprocal manner

Summary. Hepatocytes were cultured for 3 days as spheroids (aggregates) or as monolayers in basal medium and in sulfur amino acid-supplemented media. Cultured hepatocytes had low levels of cysteine dioxygenase (CDO) activity and normal levels of γ-glutamylcysteine synthetase (GCS) and cysteinesulfinate decarboxylase (CSDC) activities compared to freshly isolated cells. CDO activity increased and GCS activity decreased in a dose-response manner in cells cultured in either methionine- or cysteine-supplemented media. CSDC activity was not significantly affected by methionine supplementation. Changes in CDO and GCS were associated with changes in cysteine catabolism to taurine plus sulfate and in synthesis of glutathione, respectively. These responses are similar to those observed in liver of intact rats fed diets supplemented with sulfur amino acids. A near-maximal response of CDO or GCS activity was observed when the medium contained 1.0 mmol/L of methionine plus cyst(e)ine. Changes in CDO and GCS activities did not appear to be mediated by changes in the intracellular glutathione concentration. Cultured hepatocytes offer a useful model for further studies of cysteine metabolism and its regulation in response to sulfur amino acid availability. Received June 2, 1999/Accepted September 16, 1999     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Changes in Isozyme Profiles of Catalase,Peroxidase, and Glutathione Reductase during Acclimation to Chilling in Mesocotyls of Maize Seedlings

The response of antioxidants to acclimation and chilling in various tissues of dark-grown maize (Zea mays L.) seedlings was examined in relation to chilling tolerance and protection from chilling-induced oxidative stress. Chilling caused an accumulation of H2O2 in both the coleoptile + leaf and the mesocotyl (but not roots), and acclimation prevented this accumulation. None of the antioxidant enzymes were significantly affected by acclimation or chilling in the coleoptile + leaf or root. However, elevated levels of glutathione in acclimated seedlings may contribute to an enhanced ability to scavenge H2O2 in the coleoptile + leaf. In the mesocotyl (visibly most susceptible to chilling), catalase3 was elevated in acclimated seedlings and may represent the first line of defense from mitochondria-generated H2O2. Nine of the most prominent peroxidase isozymes were induced by acclimation, two of which were located in the cell wall, suggesting a role in lignification. Lignin content was elevated in mesocotyls of acclimated seedlings, likely improving the mechanical strength of the mesocotyl. One cytosolic glutathione reductase isozyme was greatly decreased in acclimated seedlings, whereas two others were elevated, possibly resulting in improved effectiveness of the enzyme at low temperature. When taken together, these responses to acclimation illustrate the potential ways in which chilling tolerance may be improved in preemergent maize seedlings.     

D-Aspartic acid and L-amino acids in the neural system of the amphioxus <Emphasis Type="Italic">Branchiostoma lanceolatum</Emphasis>

Summary. The lancelet (amphioxus), a cephalochordate, is the closest invertebrate relative to vertebrates, with a simple vertebrate-like body plan and a prototypical genome. We have determined D-aspartic acid (D-Asp) and major free L-amino acids (L-AAs) content in the nervous system (neural tube) of the European amphioxus Branchiostoma lanceolatum, and have compared these values with those of molluscs and human brain. The B. lanceolatum neural tube contains relatively high amounts of L-Glu, L-Asp, L-Ala and L-Gly. Thus, the amphioxus neural tube has in common with the molluscan and human nervous systems the presence of appreciable amounts of L-Glu and L-Asp, which suggests that they are the most common neurotransmitters among these phylogenetically distant animal groups. The relatively high concentration of L-Ala in amphioxus is consistent with that found in molluscs and the low concentration of taurine is consistent with that described in the human brain. The D-Asp concentration, very high in the molluscan nervous system, was rather low in amphioxus, although a little higher than the extremely low amounts observed in the human brain. Our data on free amino acids composition is in agreement with the intermediate phylogenetic position of cephalochordates, in terms of the evolutionary transition from simple to complex neural systems.     

Some aspects of the mechanism of thermal acclimation in the earthworm Lampito mauritii

Summary Inorganic ions (Ca, Mg, Na, K, Cl, SO₄) and free amino acids of the body fluids of the normal, cold and warm acclimated worms (laboratory as well as seasonal populations) are estimated. Calcium increased and chloride and sodium decreased on both cold and warm acclimation in relation to normal. But magnesium and sulphate and free amino acids increased on warm acclimation whereas potassium increased and magnesium decreased on cold acclimation. Changes in different ions in the same direction are observed in the seasonal populations. Attention is drawn to the adaptive significance of these changes in the different ions during thermal acclimation.Changes in the glycogen, RNA, protein and non-protein nitrogen, and water content in the tissues of normal and acclimated worms are studied. Glycogen increased on warm and cold acclimation, whereas RNA content, protein nitrogen and dry weight of the cold worms increased over normal. No change is observed in non-protein nitrogen on thermal acclimation. The role of these substances and the significance of the changes observed, in the operation of homeostatic mechanism compensating to temperature changes in the metabolic rate of the worms, are also discussed.Changes in the pattern of neurosecretory activity are followed with thermal acclimation and it is shown that the activity of the neurosecretory cells increased on cold and warm acclimation, but the positions of these cells, which are active, are different from normal worms in warm acclimated worms.Studies on the effect of the body fluids of acclimated worms on the tissues of normal and acclimated worms showed that the body fluids of cold acclimated worms increased the respiration of the tissues of normal and warm acclimated worms and vice-versa.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Near infrared spectroscopy, cluster and multivariate analysis hyphenated to thin layer chromatography for the analysis of amino acids

Summary. A method based on near-infrared spectroscopy (NIRS) was developed for the rapid and non-destructive determination and quantification of solid and dissolved amino acids. The statistical results obtained after optimisation of measurement conditions were evaluated on the basis of statistical parameters, Q-value (quality of calibrations), R², standard error of estimation (SEE), standard error of prediction (SEP), BIAS applying cluster and different multivariate analytical procedures. Experimental optimisation comprised the selection of the highest suitable optical thin-layer (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mm), sample temperature (10–30 °C), measurement option (light fibre, 0.5 mm optical thin-layer; boiling point tube; different types of cuvettes) and sample concentration in the range between 100 and 500 ppm. Applying the optimised conditions and a 115-QS Suprasil^? cuvette (V = 400 μl), the established qualitative model enabled to distinguish between different dissolved amino acids with a Q-value of 0.9555. Solid amino acids were investigated in the transflectance mode, allowing to differentiate them with a Q-value of 0.9155. For the qualitative and quantitative analysis of amino acids in complex matrices NIRS was established as a detection system directly onto the plate after prior separation on cellulose based thin-layer chromatography (TLC) sheets employing n-butanol, acetic acid and distilled water at a ratio of 8:4:2 (v/v/v) as an optimised mobile phase. Due to the prior separation step, the established calibration curve was found to be more stable than the one calculated from the dissolved amino acids. The found lower limit of detection was 0.01 mg/ml. Finally, this optimised TLC-NIRS method was successfully applied for the qualitative and quantitative analysis of L-lysine in apple juice. NIRS is shown not only to offer a fast, non-destructive detection tool but also to provide an easy-to-use alternative to more complicated detection methods such as mass spectrometry (MS) for qualitative and quantitative TLC analysis of amino acids in crude samples.     

Nonparticipation of extracellular glutathione in renal transport of dibasic amino acids

Microinjections of L-[14C]arginine (2.9 mM) and L-[14C]ornithine (3.4 mM) were made into renal proximal tubules of rats in the presence of methionine sulfoximine (MSO) (10, 20 mM), ATP (10 mM), and MgCl2 (20 mM) together. Absorption of both labelled amino acids dropped, respectively, by 31.1 and 49.1% compared with control microinjections. The MSO alone or ATP plus MgCl2 had no effect. These data suggest that the inhibition by MSO plus ATP plus MgCl2 is not due to direct competition between MSO and dibasic amino acids but rather to suppression of the renewal of intracellular glutathione. Such an effect is discussed in comparison with cycloleucine inhibition of dibasic amino acid transport. Addition of exogenous glutathione to microinjectates die not reverse either type of inhibition. This study shows that while intracellular glutathione may affect amino acid transport, extracellular glutathione has no effect.     

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro

Summary.  An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO₂). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39°C for 12 h with or without MSO (1 mM) or MSO₂ (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 μmol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO₂ and 2-aminobutyric acid (2AB) in B and BP. MSO₂ as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO₂; but MSO can be utilized by B and BP for producing Met. Received December 28, 2001 Accepted May 21, 2002 Published online October 14, 2002 Acknowledgements The authors are extremely grateful to Professor H. Ogawa, the University of Tokyo, Japan and Dr. Takashi Hasegawa, Miyazaki University, Japan for inserting permanent rumen fistulae in goats. We would like to thank MONBUSHO for the award of a research scholarship to Mamun M. Or-Rashid since 1996–2001. Authors' address: Shaila Wadud, Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science, Miyazaki University, Miyazaki 889-2192, Japan, Fax. +81-985-58-7201, E-mail: rafatkun@hotmail.com     

Consequences of renal mass reduction on amino acid and biogenic amine levels in nephrectomized mice

Summary. Amino acid and biogenic amine changes were investigated in nephrectomized mice ten days postsurgery. Uremic mice exhibited changes in amino acid concentrations in plasma, urine and brain. Particularly plasma methionine, citrulline and arginine levels were significantly enhanced in nephrectomized mice compared to controls whereas serine was decreased. Urinary excretion of methionine, citrulline and alanine was higher in nephrectomized mice compared to controls whereas many amino acids were increased in brain of nephrectomized mice. Brain and urinary amino acid changes were more pronounced in the 75% than in the 50% nephrectomized mice. Brain norepinephrine and dopamine and its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly increased whereas serotonin was decreased comparing the 75% nephrectomized mice to the sham-operated mice. This study demonstrates that at very early stages of renal insufficiency, specific amino acid and biogenic amine changes occur in plasma, urine and brain. These alterations might depend qualitatively and quantitatively on the degree of functional renal mass reduction. Received April 5, 1999     

Effects of D-amino acids on lipoperoxidation in rat liver and kidney mitochondria

Summary. The effects of the amino acids D-ser, D-asp, and D-ala on lipoperoxidation under conditions of hypertension, alcoholism, and ammonemia in rat liver and kidney mitochondria were studied. Under normal conditions, D-alanine increased in 54% free radicals production in liver mitochondria (p < 0.05). The D-amino acids had no effect on kidney mitochondria. D-ser and D-ala increased lipoperoxidation in spontaneously hypertensive rats (SHR) as compared with their normotensive genetic control Wistar-Kyoto (WKY) rats (p < 0.05). During hypertension and in oxidative stress in the presence of calcium, only D-ala produced 46% and 29% free radicals in liver and kidney mitochondria (p < 0.05), respectively. During chronic alcoholism, D-ser increased lipoperoxidation in 80% in kidney mitochondria (p < 0.05), as compared to control. During ammonemia, D-ser produced 41% free radicals.     

Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5 h with [U-¹³C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH₄Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.     

Inhibition of glutamine synthetase triggers apoptosis in asparaginase-resistant cells.

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24 h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.